Project Summary A simple point-of-care (POC) diagnostic test, with laboratory-level performance and the ability to detect the virus across the active phase of infection, is vital to efficient prevention and response efforts during the current Zika virus outbreak, and future management of the disease as it spreads. The ability to differentiate between Zika and Dengue viruses (ZIKV, DENV) is also of utmost importance, particularly in low-resource settings where Dengue is endemic. While lateral flow immunoassays (LFIs) to detect IgM antibodies are deployable, they cannot detect early infection (0-7 days post-onset of symptoms, DPO). On the other hand, the lack of infrastructure and trained lab staff prevents wide use of highly sensitive nucleic acid tests (NATs). There is a need for a simple POC test that is highly sensitive and can detect infection during the entire period of active infection (0-90 DPO). Daktari proposes to develop a simple POC ZIKV/DENV Ag/Ab combo test sensitive and specific enough to detect and differentiate Zika and Dengue infections. By combining detection of viral antigen with host IgM antibodies, we extend the use window of the test to the entire active infection period. Non-structural 1 (NS1) antigen has been validated as a biomarker for arboviruses and NS1 antigen tests exist for Dengue; their sensitivity, however, is poor when implemented at the POC. No Zika NS1 Ag tests have been developed to date. In this Fast-Track program, we will adapt Daktari's POC Virus and Protein (ViPr) platform to detect ZIKV and DENV NS1 and IgM in blood. ViPr consists of (1) Daktari's core technology for automated microfluidic sample preparation (load 25-100 ?L of fingerprick blood into the cartridge - insert cartridge into instrument); (2) a high-sensitivity immunoassay detection technology based on silver nanoparticle labels and anodic stripping voltammetry, proven to enable limits of detection (LOD) ?1 pg/mL protein in plasma (3 logs better than LFAs); and (3) a simple, robust instrument with built-in connectivity and a self-contained disposable cartridge. We will first demonstrate the feasibility of adapting Daktari's viral protein detection assay to the detection of Zika and Dengue antigens. Phase I will focus on developing and screening high-affinity and specific antibodies against the non-structural 1 (NS1) antigens of ZIKV and DENV. We will then conjugate selected antibodies to the magnetic and silver particles, and we will optimize the assay protocol. Finally, we will evaluate assay performance in plasma samples. In Phase II, we will develop the Daktari ViPr IgM assay, and then adapt the ViPr cartridge to implement the ZIKV/DENV NS1 and IgM assays. We will also upgrade the Daktari instrument to incorporate enough actuators to perform the four parallel assays. Finally, performance of the integrated test will be evaluated in clinical samples. With the simple-to-use and sensitive Daktari ZIKV/DENV Ab/Ag combo system, it will be possible for health workers with minimal training to diagnose active Zika infection quickly and accurately, anywhere in the world.